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A A Kossiakoff

Publications and source records attributed to A A Kossiakoff.

At least 19 recordsLinked to original sources
PubMed1992

Human growth hormone and extracellular domain of its receptor: crystal structure of the complex.

Binding of human growth hormone (hGH) to its receptor is required for regulation of normal human growth and development. Examination of the 2.8 angstrom crystal structure of the complex between the hormone and the extracellular domain of its receptor (hGHbp) showed that the complex consists of one molecule of growth hormone per two molecules of receptor. The hormone is a four-helix bundle with an unusual topology. The binding protein contains two distinct domains, similar in some respects to immunoglobulin domains. The relative orientation of these domains differs from that found between constant and variable domains in immunoglobulin Fab fragments. Both hGHbp domains contribute residues that participate in hGH binding. In the complex both receptors donate essentially the same residues to interact with the hormone, even though the two binding sites on hGH have no structural similarity. Generally, the hormone-receptor interfaces match those identified by previous mutational analyses. In addition to the hormone-receptor interfaces, there is also a substantial contact surface between the carboxyl-terminal domains of the receptors. The relative extents of the contact areas support a sequential mechanism for dimerization that may be crucial for signal transduction.

Binding Sites↗
PubMed1992

Crystal structure of the kringle 2 domain of tissue plasminogen activator at 2.4-A resolution.

The crystal structure of the kringle 2 domain of tissue plasminogen activator was determined and refined at a resolution of 2.43 A. The overall fold of the molecule is similar to that of prothrombin kringle 1 and plasminogen kringle 4; however, there are differences in the lysine binding pocket, and two looping regions, which include insertions in kringle 2, take on very different conformations. Based on a comparison of the overall structural homology between kringle 2 and kringle 4, a new sequence alignment for kringle domains is proposed that results in a division of kringle domains into two groups, consistent with their proposed evolutionary relation. The crystal structure shows a strong interaction between a lysine residue of one molecule and the lysine/fibrin binding pocket of a noncrystallographically related neighbor. This interaction represents a good model of a bound protein ligand and is the first such ligand that has been observed in a kringle binding pocket. The structure shows an intricate network of interactions both among the binding pocket residues and between binding pocket residues and the lysine ligand. A lysine side chain is identified as the positively charged group positioned to interact with the carboxylate of lysine and lysine analogue ligands. In addition, a chloride ion is located in the kringle-kringle interface and contributes to the observed interaction between kringle molecules.

Amino Acid Sequence↗
PubMed1992

Solvent structure in crystals of trypsin determined by X-ray and neutron diffraction.

The solvent structure in orthorhombic crystals of bovine trypsin has been independently determined by X-ray diffraction to 1.35 A resolution and by neutron diffraction to 2.1 A resolution. A consensus model of the water molecule positions was obtained using oxygen positions identified in the electron density map determined by X-ray diffraction, which were verified by comparison to D2O-H2O difference neutron scattering density. Six of 184 water molecules in the X-ray structure, all with B-factors greater than 50 A2, were found to be spurious after comparison with neutron results. Roughly two-thirds of the water of hydration expected from thermodynamic data for proteins was localized by neutron diffraction; approximately one-half of the water of hydration was located by X-ray diffraction. Polar regions of the protein are well hydrated, and significant D2O-H2O difference density is seen for a small number of water molecules in a second shell of hydration. Hydrogen bond lengths and angles calculated from unconstrained refinement of water positions are distributed about values typically seen in small molecule structures. Solvent models found in seven other bovine trypsin and trypsinogen and rat trypsin structures determined by X-ray diffraction were compared. Internal water molecules are well conserved in all trypsin structures including anionic rat trypsin, which is 65% homologous to bovine trypsin. Of the 22 conserved waters in trypsin, 19 were also found in trypsinogen, suggesting that they are located in regions of the apoprotein that are structurally conserved in the transition to the mature protein. Seven waters were displaced upon activation of trypsinogen. Water structure at crystal contacts is not generally conserved in different crystal forms. Three groups of integral structural water molecules are highly conserved in all solvent structures, including a spline of water molecules inserted between two beta-strands, which may resemble an intermediate in the formation of beta sheets during the folding of a protein.

Deuterium↗
PubMed1992

Analysis of solvent structure in proteins using neutron D2O-H2O solvent maps: pattern of primary and secondary hydration of trypsin.

A method of determining the water structure in protein crystals is described using neutron solvent difference maps. These maps are obtained by comparing the changes in diffracted intensities between two data sets, one in which H2O is the major solvent constituent, and a second in which D2O is the solvent medium. To a good first approximation, the protein atom contributions to the scattering intensities in both data sets are equal and cancel, but since H2O and D2O have very different neutron-scattering properties, their differences are accentuated to reveal an accurate representation of the solvent structure. The method also employs a series of density modification steps that impose known physical constraints on the density distribution function in the unit cell by making real space modifications directly to the density maps. Important attributes of the method are that (1) it is less subjective in the assignment of water positions than X-ray analysis; (2) there is threefold improvement in the signal-to-noise ratio for the solvent density; and (3) the iterative density modification produces a low-biased representation of the solvent density. Tests showed that water molecules with as low as 10% occupancy could be confidently assigned. About 300 water sites were assigned for trypsin from the refined solvent density; 140 of these sites were defined in the maps as discrete peaks, while the remaining were found within less-ordered channels of density. There is a very good correspondence between the sites in the primary hydration layer and waters found in the X-ray structure. Most water sites are clustered into H-bonding networks, many of which are found along intermolecular contact zones. The bound water is equally distributed between contacting apolar and polar atoms at the protein interface. A common occurrence at hydrophobic surfaces is that apolar atoms are circumvented by one or more waters that are part of a larger water network. When the effects on surface accessibility by neighboring molecules in the crystal lattice are taken into consideration, only about 29% of the surface does not interface ordered water. About 25% of the ordered water is found in the second hydration sphere. In many instances these waters bridge larger clusters of primary layer waters. It is apparent that, in certain regions of the crystal, the organization of ordered water reflects the characteristics of the crystal environment more than those of trypsin's surface alone.

Deuterium↗
PubMed1992

Variability of conformations at crystal contacts in BPTI represent true low-energy structures: correspondence among lattice packing and molecular dynamics structures.

The structures of five basic pancreatic trypsin inhibitor (BPTI) molecules are compared to establish the extent and nature of the conformational variability resulting from crystal packing effects. BPTI is an ideal system to evaluate such factors because of the availability of high resolution X-ray models of five different BPTI structures, each in a different crystal packing environment. Differences observed among the structures are found to be distributed throughout the molecule, although the regions that display most variability are associated with the loop structures (residues 14-17 and 24-29). The regions of structure that show the largest rms deviations from the mean of the five packing motifs correlate well with the presence of intermolecular contacts in the crystal lattice. For most of the molecules there is also a correspondence between a larger number of intermolecular contacts and systematically higher B-factors, although it is not apparent whether this is induced by the crystal contact or results from the fact that the contacts are made predominantly through surface loops. The conformational differences seen among the X-ray models constitute more than local shifts at the lattice contact surfaces, and in fact involve in some cases the making and breaking of intramolecular H-bonds. The magnitudes of the differences among packing models are significantly larger than those usually associated with changes induced by mutagenesis; for instance; the structural differences at the site of mutation observed on removing an internal disulfide from the molecule are significantly less than those associated with lattice contact effects. The crystal packing conformations are compared to representative structures of BPTI generated during a 96-psec molecular dynamics (MD) simulation. This comparison shows a high level of correspondence between the protein flexibility indicated by the X-ray and MD analyses, and specifically between those regions that are most variable. This suggests that the regions that show most variability among the crystal packing models are not artifacts of crystallization, but rather represent true low-energy conformers that have been preferentially selected by crystallization factors.

Amino Acid Sequence↗
PubMed1992

Structural effects induced by mutagenesis affected by crystal packing factors: the structure of a 30-51 disulfide mutant of basic pancreatic trypsin inhibitor.

The X-ray structure of the C30V/C51A disulfide mutant of basic pancreatic trypsin inhibitor (BPTI) has been analyzed at 1.6 A resolution. The mutant crystallizes in a cell having two molecules in the asymmetric unit. The packing environments of these two molecules are quite different, allowing for an assessment of which among the observed structural changes result from the mutation and which are produced by lattice packing considerations. The removal of the 30-51 disulfide bridge has little apparent affect on the B-factors of segments of adjacent polypeptide chain, although there are distinct differences in the structure compared to wild-type BPTI crystal structures. Both of the two C30V/C51A molecules show differences at the mutation site when compared to another 30-51 disulfide mutant, C30A/C51A, presumably due to the larger steric bulk of a valine versus an alanine at residue 30. A comparison of the two independent C30V/C51A molecules indicates that there are significant differences between them even at the site of mutation. The description of the specific structural differences of each molecule differs in detail and suggests different conclusions about the nature of structural perturbation near 30-51. In addition, when these two molecules are compared to two different wild-type structures, which had been determined from different space groups, a somewhat different pattern of changes is observed. These findings indicate that crystal packing can influence the observed perturbations in mutant structures.

Alanine↗
PubMed1992

Structural consequences of mutation.

The way a protein responds to mutation provides key insights into its architecture and energetics. Mutations are improving the understanding both of protein folding and stability, and of the adaptability of the hydrophobic core. The importance of intermolecular effects in crystal structures is being emphasized and new insights into the correspondence between crystal and solution structures are being developed.

Mutation↗
PubMed1991

Crystals of the complex between human growth hormone and the extracellular domain of its receptor.

Single crystals suitable for high-resolution diffraction studies have been grown of the human growth hormone (hGH) complexed to the extracellular domain of its cloned receptor from the human liver (hGHbp), using the technique of repeat seeding. The crystals are in space group P2(1)2(1)2, with a = 145.8 A, b = 68.6 A, c = 76.0 A, and diffract to at least 2.7 A resolution on a rotating anode X-ray source. Analysis of the composition of these crystals showed the stoichiometry of the complex to be hGH: (hGHbp)2. This finding, coupled with biochemical data on the complex in solution, indicates that the biologically significant dimerization of the growth hormone receptor is mediated through a single hormone molecule. Structure determination of the complex is currently being completed.

Crystallization↗
PubMed1991

X-ray structure of human relaxin at 1.5 A. Comparison to insulin and implications for receptor binding determinants.

The X-ray crystal structure of relaxin at 1.5 A resolution is reported for the physiologically active form of the human hormone. Relaxin is a small, two-chain polypeptide that is a member of the protein hormone family that also includes insulin and the insulin-like growth factors IGF-I and IGF-II. These hormones have biologically diverse activities but are structurally similar, sharing a distinctive pattern of cysteine and glycine residues. The predicted structural homology of relaxin to insulin is confirmed by this structural analysis; however, there are significant differences in the terminal regions of the b-chain. Although relaxin, like insulin, crystallizes as a dimer, the orientation of the molecules in the respective dimers is completely different. The region of the relaxin molecule proposed to be involved in receptor binding is part of the dimer interface, suggesting that some of the other residues contained in the dimer contact surface might be receptor binding determinants as well. The proposed receptor binding determinants for insulin likewise include residues at its dimer interface. However, because the dimer contacts of relaxin and insulin are quite different, it appears that these two structurally related hormones have evolved somewhat dissimilar mechanisms for receptor binding.

Amino Acid Sequence↗
PubMed1991

Neutron structure of subtilisin BPN': effects of chemical environment on hydrogen-bonding geometries and the pattern of hydrogen-deuterium exchange in secondary structure elements.

The neutron structure of subtilisin BPN' has been refined and analyzed at 2.0-A resolution. The structure studied was a mutant variant of subtilisin, Met222----Gln, and was used because large, uninhibited crystals could be grown, which was not the case for the native molecule. Comparison of the structure with that of the native molecule indicated that the two structures are essentially the same. Using the capability of the neutron method to locate hydrogen and deuterium atoms, the protonation states of the six histidine residues were assigned. The active site histidine, His64, was found to be neutral at the pH of the analysis (pH 6.1). This group has an unexpectedly low pKa compared to assignments made by other techniques. The altered pKa of the group could result from electrostatic effects of other molecules in the crystal lattice. The dihedral conformations of a majority of the hydroxyl rotors were assigned. The preferred orientation was trans (180 degrees) with the other two low-energy conformers (60 degrees, 300 degrees) about equally populated. For the serines, about 21% of the hydroxyls act exclusively as H-bond acceptors and 37% as H-bond donors, and in 42% the group functions as both. The experimentally observed dihedral conformations were compared to predicted conformations based on calculated energy criteria and showed a strong correspondence. Deviation from low-energy states could usually be explained by local electrostatic effects. The hydrogen exchange pattern of subtilisin identified the beta-sheet and alpha-helix secondary structure elements to be the most resistant to exchange. Fifty-five percent of the peptide amide hydrogens were fully exchanged, 15% unexchanged, and 30% partially exchanged. The largest concentration of unexchanged sites was in the seven-stranded parallel beta-sheet, in which there were 11 fully protected groups. Little correlation was found between H-bond length and angle and a peptide group's susceptibility toward exchange. Of the five alpha-helices the most protected from exchange is the one defined by residues 224-236. The pattern of exchange identifies regions in this helix where the H-bonding regularity is disrupted.

Binding Sites↗
PubMed1990

X-ray crystal structure of the protease inhibitor domain of Alzheimer's amyloid beta-protein precursor.

Alzheimer's amyloid beta-protein precursor contains a Kunitz protease inhibitor domain (APPI) potentially involved in proteolytic events leading to cerebral amyloid deposition. To facilitate the identification of the physiological target of the inhibitor, the crystal structure of APPI has been determined and refined to 1.5-A resolution. Sequences in the inhibitor-protease interface of the correct protease target will reflect the molecular details of the APPI structure. While the overall tertiary fold of APPI is very similar to that of the Kunitz inhibitor BPTI, a significant rearrangement occurs in the backbone conformation of one of the two protease binding loops. A number of Kunitz inhibitors have similar loop sequences, indicating the structural alteration is conserved and potentially an important determinant of inhibitor specificity. In a separate region of the protease binding loops, APPI side chains Met-17 and Phe-34 create an exposed hydrophobic surface in place of Arg-17 and Val-34 in BPTI. The restriction this change places on protease target sequences is seen when the structure of APPI is superimposed on BPTI complexed to serine proteases, where the hydrophobic surface of APPI faces a complementary group of nonpolar side chains on kallikrein A versus polar side chains on trypsin.

Alzheimer Disease↗
PubMed1990

Crystal structures of subtilisin BPN' variants containing disulfide bonds and cavities: concerted structural rearrangements induced by mutagenesis.

The X-ray structures of four genetically engineered disulfide variants of subtilisin have been analyzed to determine the energetic and structural constraints involved in inserting disulfide bonds into proteins. Each of the engineered disulfides exhibited atypical sets of dihedral angles compared with known structures of natural disulfide bridges in proteins and affected its local structural environment to a different extent. The disulfides located in buried regions, Cys26-Cys232 and Cys29-Cys119, induced larger changes than did Cys24-Cys87 and Cys22-Cys87, which are located on the surface of the molecule. An analysis of the concerted changes in secondary structure units such as alpha-helices and beta-sheets indicated systematic long-range effects. The observed changes in the mutants were largely distributed asymmetrically around the inserted disulfides, reflecting different degrees of inherent flexibility of neighboring secondary structure types. The disulfide substitution in each variant molecule created some invaginations or cavities, causing a reorganization of the surrounding water structure. These changes are described, as well as the changes in side chain positions of groups that border the cavities.

Base Sequence↗
PubMed1990

Hydroxyl hydrogen conformations in trypsin determined by the neutron diffraction solvent difference map method: relative importance of steric and electrostatic factors in defining hydrogen-bonding geometries.

Neutron diffraction maps have been used to assign the rotor conformations of the hydroxyl hydrogens in trypsin. Knowledge of these conformations is used to assess the relative importance of steric and electrostatic effects in conferring the H-bonding geometries of these groups. A general finding was that most hydroxyl groups are rotationally ordered with their highest populated conformation near the low-energy staggered orientation. For the low-energy conformers (-60 degrees, 60 degrees, 180 degrees) of serine and threonine, the trans (-180 degrees) position is most highly populated followed by +60 degrees. In trypsin, only 1 of 24 serines was found in the -60 degrees conformer. Serine hydroxyls preferentially act as H-bond acceptors and rarely are observed as H-bond donors alone. Threonines were found to be more likely than serines to participate in two H bonds; tryosines were found to prefer to act as donors. In H-bonding situations in which there was incompatibility between the energies defining the barrier to rotation and the local electrostatics, the electrostatic criteria dominated. Overall, the findings support a model of H bonding where there exists strong inherent complementarity between the low-energy hydroxyl orientations and the local electrostatic environment.

Hydrogen Bonding↗
PubMed1990

Structural effects induced by removal of a disulfide-bridge: the X-ray structure of the C30A/C51A mutant of basic pancreatic trypsin inhibitor at 1.6 A.

The X-ray structure of a variant of basic pancreatic trypsin inhibitor (BPTI) has been analyzed to determine the structural accommodation resulting from removal of a disulfide cross-link in a protein. The disulfide removed, Cys30-Cys51, has been implicated in both the folding pathway of the protein and its overall thermal stability. In the variant studied, C30A/C51A, the disulfide cysteines were replaced by less bulky alanines. The atomic displacements observed for C30A/C51A indicate a set of concerted shifts of two segments of chains, which together significantly diminish a packing defect at the site of the removed cysteine sulfur atoms. The observed structural changes are distributed asymmetrically around the sites of mutation, indicating that the adjacent beta-sheet is more resistant to the perturbation than the alpha-helix on the opposite side of the disulfide bond. The thermal parameters of groups involved in the structural accommodation are not significantly altered. A comparison of the X-ray structures reported for native BPTI determined in three different crystal forms indicates that the magnitude of its conformational variability exceeds that of the structural changes caused by the disulfide removal. This emphasizes the necessity of using isomorphous crystal systems to determine the relatively small effects due to mutation.

Aprotinin↗
PubMed1988

Tertiary structure is a principal determinant to protein deamidation.

The protein deamidation process involves the conversion of the amide side-chain moieties of asparagine and glutamine residues to carboxyl groups. This conversion is an unusual form of protein modification in that it requires catalysis by an intramolecular reaction where both the substrate (asparagine and glutamine side chains) and "catalytic site" (the peptide nitrogen of the succeeding residue) are constituents of several consecutive residues along the polypeptide chain. The stereochemical factors governing this process were studied with a data base derived from the neutron crystallographic structure of trypsin from which amide groups and oxygen can be unambiguously differentiated because of their different neutron scattering properties. The neutron structure allowed for the direct determination of those residues that were deamidated; 3 of 13 asparagine residues were found to be modified. These modified residues were clearly distinguished by a distinct local conformation and hydrogen-bonding structure in contrast to those observed for the other asparagine residues. No correlation was found between preference to deamidate and the chemical character of residues flanking the site, as had been proposed from previous peptide studies.

Amides↗
PubMed1977

Structure of bovine trypsinogen at 1.9 A resolution.

The three-dimensional crystal structure of bovine trypsinogen at approximately pH 7.5 was initially solved at 2.6 A resolution using the multiple isomorphous replacement method. Preliminary refinement cycles of the atomic coordinates trypsinogen have been carried out first to a resolution of 2.1 A, and later to 1.9 A, using constrained difference Fourier refinement; During the process, structure factors Fc and phi c were calculated from the trypsinogen structure and final interpretation was based on an electron-density map computed with terms (2 Fo - Fc) and phases phic at a resolution of 1.9 A. Crystals of trypsinogen grown from ethanol-water mixtures are trigonal with space group P3121, and cell dimension a = 55.17 A and c = 109.25 A. The structure is compared with the bovine diisopropylphosphoryltrypsin structure at approximately pH 7.2, oirginally determined from orthohombic crystals by Stroud et al. (Stroud, R.M., Kay L.M., and Dickerson, R.E. (1971), Cold Spring Harbor Symp. Quant. Biol. 36, 125-140; Stroud, R.M., Kay, L.M., and Dickerson, R.E. (1974), J. Mol. Biol. 83, 185-208), and later refined at 1.5 A resolution by Chambers and Stroud (Chambers, J.L., and Stroud, R.M. (1976), Acta Crystallogr. (in press)). At lower pH, 4.0-5.5 diogen, with cell dimensions a = 55.05 A and c = 109.45 A. This finding was used in the solution of the six trypsinogen heavy-atom derivatives prior to isomorphous phase analysis, and as a further basis of comparison between trypsinogen and the low pH trypsin structure. There are small differences between the two diisopropylphosphoryltrypsin structures. Bovine trypsinogen has a large and accessible cavity at the site where the native enzyme binds specific side chains of a substrate. The conformation and stability of the binding site differ from that found in trypsin at approximately pH 7.5, and from that in the low pH form of diisopropylphosphoryltrypsin. The catalytic site containing Asp-102, His-57, and Ser-195 is similar to that found in trypsin and contains a similar hydrogen-bounded network. The carboxyl group of Asp-194, which is salt bridged to the amino terminal of Ile-16 in native trypsin or other serine proteases, is apparently hydrogen bonded to internal solvent molecules in a loosely organized part of the zymogen structure. The unusually charged N-terminal hexapeptide of trypsinogen, whose removal leads to activation of the zymogen, lies on the outside surface of the molecule. There are significant structural changes which accompany activation in neighboring regions, which include residues 142-152, 215-550, 188A-195. The NH group of Gly-193, normally involved in stabilization of reaction intermediates (Steitz, T.A., Henderson, R., and Blow, D.M. (1969), J. Mol. Biol. 46, 337-348; Henderson, R. (1970), J. Mol. Biol. 54, 341-354; robertus, J.D., Kraut, J., Alden, R.A., and Birkoft, J.J. (1972), Biochemistry 11, 4293-4303) in the enzyme, is moved 1.9 A away from its position in trypsin...

Animals↗